Anhydrous silver dihydrogen citrate compositions

ABSTRACT

Anhydrous silver dihydrogen citrate compositions comprise silver dihydrogen citrate and citric acid. The anhydrous compositions can be prepared by freeze-drying. The anhydrous compositions can be reconstituted with a suitable diluent to form silver dihydrogen citrate compositions. The anhydrous compositions can be reconstituted and applied to a variety of substrates to impart an antimicrobial effect on the substrates.

This invention claims priority from U.S. provisional application Ser.No. 60/498,347, filed Aug. 28, 2003, which is incorporated herein byreference in its entirety.

FIELD OF THE INVENTION

This invention generally relates to antimicrobial compositions. Inparticular, the invention provides compositions comprising anhydroussilver dihydrogen citrate and citric acid, processes of making thecompositions and antimicrobial methods of using the solid compositions.

BACKGROUND OF THE INVENTION

Antimicrobial metal ion solutions have been used as disinfectants.Silver solutions have been used as disinfectants in cooling towers,swimming pools, hot water systems in hospitals, portable water systemsand spa pools. Additionally, silver ion solutions have been prepared forthe treatment of wounds, however the silver ions used in the proposedmethods are unstable and must be generated near the wound in order todeliver a therapeutic dose to the wound site.

Silver salts, such as silver citrate salts, have also been proposed asantimicrobial dusting agents. However, these dusting agents must be keptdry and are generally not convenient for imparting preservative value toconsumer products or for delivering antimicrobial effects to an end useror to the environment of the end user. Colloidal silver has found avariety of uses, including: as a wood preservative; as a disinfectant offood and beverage containers and industrial processing equipment; as abactericide in paints; as a biocide in synthetic polymer films; and as asterilizing agent in bandages.

Aqueous solutions of silver dihydrogen citrate and citric acid have beendisclosed in U.S. Pat. No. 6,197,814 (incorporated herein in itsentirety) as disinfectants in a variety of settings. These watersolutions of silver dihydrogen citrate and citric acid are made bypassing an electrical current through a pair of silver electrodes thatare immersed in a water solution of citric acid. These silver dihydrogencitrate solutions are effective against a wide-variety of microbes,including bacteria, viruses and fungi, and are non-toxic in the humanenvironment at concentrations effective to combat microbial infestation.However, aqueous solutions are bulky and heavy, and are thusuneconomical to store and to transport.

There is a need for silver dihydrogen citrate compositions that can beeconomically stored, transported or both. Such compositions should besubstantially free of water in order to minimize the space required fortheir storage, as well to minimize their shipping weight. Suchcompositions should confer the same beneficial antimicrobial effect assolutions of silver dihydrogen citrate when combined with water or otheraqueous solution. The present invention satisfies this need and providesrelated advantages as well.

SUMMARY OF THE INVENTION

The present invention provides compositions comprising anhydrous silverdihydrogen citrate and citric acid and a process of making saidcompositions. The process includes freezing a stock solution of aqueoussilver dihydrogen citrate and then freeze-drying the frozen stocksolution to produce anhydrous silver dihydrogen citrate.

The invention further provides a method of using anhydrous silverdihydrogen citrate and citric acid by combining it with an aqueousdiluent to produce an antimicrobially effective aqueous silverdihydrogen citrate solution.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides compositions comprising anhydrous silverdihydrogen citrate and citric acid. Surprisingly, it has been found thatthe anhydrous silver dihydrogen citrate compositions may bereconstituted after a period of time to make stable silver dihydrogencitrate solutions having effective antimicrobial activity. The anhydrouscompositions offer the advantage of ease and economy of storage. Theyalso offer the advantage of being more economical to transport thanaqueous solutions of silver dihydrogen citrate. As the anhydrouscompositions may be conveniently reconstituted with water or anotheraqueous diluent, the anhydrous compositions offer a convenientalternative to aqueous solutions of silver dihydrogen citrate.

Silver dihydrogen citrate has been identified as the activeantimicrobial ingredient in a solution produced by electrolysis ofsilver electrodes immersed in a citric acid electrolyte solution. Thestructure of silver dihydrogen citrate can be represented by formula I:

wherein Ag⁺ is silver ion and the negatively charged carbonate (COO⁻) isthe most likely charged group in the dihydrogen citrate moiety. Thechemical formula for this complex is AgC₆H₇O₇, and its molecular weightis 298.99 g/mol. As can be seen from the formula above, silverdihydrogen citrate is a salt, wherein the silver ion is the cation andthe dihydrogen citrate ion is the anion. In solution, the salt ispresent in a dissociated state, the cation and anion being surrounded bywater molecules, which associate with the ions through their polar OHgroups.

Citric acid can be represented by formula II:

having a chemical formula of C₆H₈O₇ and a molecular weight of 192.13g/mol. Citric acid is readily available from commercial sources. Citricacid is very soluble in water. Aqueous solutions of citric acid may beup to about 60% (wt/vol) citric acid in water at 25° C.

The invention also provides methods of making solid silver dihydrogencitrate compositions. The method starts with providing a stock solutionof silver dihydrogen citrate, as described in U.S. Pat. No. 6,197,814. Asummary of the process of making the silver dihydrogen citrate solutionis as follows.

A process of making the stock solution of silver dihydrogen citrate isan electrolytic process. The process begins with preparation of anelectrolyte solution, which is an aqueous solution comprising citricacid. It has been found that citric acid solutions having citric acidconcentrations in the range of about 1% (wt./vol.) to about thesolubility limit of citric acid in water (about 60% wt./vol.) aresuitable for preparing silver dihydrogen citrate solutions. In someembodiments, the range is about 5 to about 25 wt % citric acid, withspecific values being 10, 15 and 20 wt %. The electrolyte may compriseother ingredients that will not interfere with the electrolytic process.

A pair of silver electrodes are immersed into the electrolyte solutionat a suitable spacing to allow ionic current flow between them. Asuitable spacing is greater than about 2 mm, for example from about 2 toabout 8 mm. An electrolytic potential is applied across the electrodesto create an ionic current flow between the electrodes. While it ispossible to use a D.C. power supply to apply the electrolytic potential,it has been found desirable to apply an alternating wave form to theelectrodes. A suitable voltage is about 12 to about 50 volts. A suitablealternating waveform applies a pulsed potential having a peak voltage ofabout 12 to 50 volts to the electrodes, with periodic intermittent phasereversal. The resulting flow of ions through the electrolyte solutionresults in production of an aqueous solution of silver dihydrogencitrate and citric acid. This solution may be further processed, forexample by settling, filtration or both, before being used in theprocess of making anhydrous silver dihydrogen citrate.

It is possible to perform the foregoing process batch-wise or in acontinuous process. It is also possible to recirculate silver dihydrogencitrate solution through the electrolytic cell to increase the finalconcentration of silver dihydrogen citrate in the solution. The personof skill in the art will recognize that the foregoing steps may bepracticed in a number of different variations known in the art. Theprocess is described in filler detail in U.S. Pat. No. 6,197,814(incorporated by reference) and in the examples below. The resultingsolution is referred to herein as the “stock solution,” meaning a watersolution of silver dihydrogen citrate and citric acid that has not yetbeen subjected to freeze-drying.

Having provided the stock solution, the next step is to remove waterfrom the stock solution to produce anhydrous silver dihydrogen citrate.It has been found that freeze-drying results in a superior product. Inthe freeze-drying process, a frozen solution comprising silverdihydrogen citrate and citric acid solution is subjected to a vacuum,whereby the temperature and pressure of the solution are reduced belowthe triple point of water. Below the triple point, water will sublimate,passing directly from the solid phase into the gaseous phase. In someembodiments, the solution is first frozen, then placed in afreeze-drying apparatus. The solution may be frozen, for example, byplacing it in a cold environment such as a freezer. In otherembodiments, the solution is placed in the freeze-drying apparatus andthen frozen. As evaporation is an endothermic process, the solution maybe frozen by placing it in the freeze-drying apparatus and applying thevacuum. As water evaporates from the solution sample, it draws heat fromthe sample, thereby lowering the sample's temperature to below thetriple point of the sample. In such cases, it is useful to takemeasures, such as centrifugation, to prevent foaming of the sample. Itis also useful to heat the sample during the freeze-drying process inorder to compensate for the latent heat deficit of the frozen solutioncaused by sublimation of water. The person skilled in the art willrecognize that heating the frozen sample will generally speed thefreeze-drying process, so long as the sample's temperature remains belowthe triple point of the solution.

Freeze drying apparatuses are known and available from a number ofsuppliers; or they may be constructed using procedures known in the art.A freeze-drying apparatus comprises a drying chamber, a condenser totrap water of sublimation, a cooling system that supplies refrigerant tothe vacuum chamber and the condenser, a vacuum system to apply a vacuumto the drying chamber. The freeze-drying apparatus may also have atemperature control device that allows one to cool the sample to thefreezing point and to heat the sample to speed the sublimation process.The temperature control device will in some cases include refrigerationcoils that circulate refrigerant. The temperature control device mayalso include heating coils or another appropriate device to heat thesample during the freeze-drying process.

The freeze-drying process removes sufficient water to form an anhydrouscomposition comprising silver dihydrogen citrate and citric acid. Theanhydrous silver dihydrogen citrate crystals are translucent-gray incolor. In some embodiments, these crystals are ground to a fine powderand stored for an indefinite period of time until they arereconstituted. In some embodiments, the powder is stored in alight-proof container, such as an opaque bottle or in a light-proof boxto prevent decomposition by light.

Surprisingly it has been found that a silver dihydrogen citratecomposition reconstituted using the same volume of diluent, such aswater, as was present in the stock solution possesses the sameconcentration of free silver ion as was in the stock solution, withoutany noticeable degradation, reduction of the redox state of silver, orformation of colloid. As silver ion is the active antimicrobial agent ina silver dihydrogen citrate composition, such reconstituted compositionsare expected to possess the same antimicrobial properties as the stocksolution. The reconstituted compositions may thus be used in any mannerthat the stock solutions could have been used.

The term “composition comprising anhydrous silver dihydrogen citrate andcitric acid” means a solid composition containing silver dihydrogencitrate and citric acid. Thus, while the composition may containdetectable quantities of water, it does not contain enough water for thecomposition to be fluid. While the solid composition may be amorphous orcrystalline, a crystalline form is especially desirable, as such isformed by the freeze-drying process described, and is consideredespecially stable. Thus, the invention further provides crystallinecompositions comprising anhydrous silver dihydrogen citrate and citricacid.

The invention further provides anhydrous silver dihydrogen citratecompositions that are substantially free of water. The term“substantially free” means that the sample comprises less than the lowerdetectable limit of water. While it is theoretically possible to removeall water from the sample, in some cases it is sufficient to dry thesample to the point that the amount of remaining water, if any, is belowthe detection limit for art-recognized detection means. Some detectionmeans that may be mentioned are gas chromatography (GC), nuclearmagnetic resonance spectrometry (NMR), mass spectrometry (MS), orcombinations of two or more thereof, for example GC-MS.

The invention further provides anhydrous silver dihydrogen citratecontaining an amount of water above the detection limit of water byconventional testing processes. In such embodiments, it is preferredthat the anhydrous silver dihydrogen citrate comprises less than about2%, in particular less than about 1%, more particularly less than about0.5% and even more particularly less than about 0.1% water (wt./wt. %);The person skilled in the art will recognize that such anhydrouscompositions may be obtained by performing the freeze-drying processunder conditions, such as vacuum, length of time and heating, sufficientto remove the appropriate amount of water from the frozen sample.

The invention further provides compositions comprising anhydrous silverdihydrogen citrate and a molar excess of citric acid. The term “molarexcess” means an amount of citric acid such that, for each mole ofsilver dihydrogen citrate in the composition, there is greater than onemole of citric acid. For example, a 1-60% wt/vol solution of citric acidthat has been subjected to electrolysis as previously describe willcontain from about 0.00001 to about 1 wt % silver ion. In a particularexample, a 10% citric acid solution treated by the electrolysis processdescribed above contains about 0.1% silver ion. The person of skill inthe art will recognize that there is a molar excess of citric acid tosilver dihydrogen citrate in the solution. In this example, the molarexcess is about 15 fold. The person of skill in the art will furtherrecognize that, as citric acid is non-volatile, the solid silverdihydrogen citrate composition produced by the foregoing freeze-dryingprocess will contain a molar excess of citric acid over silverdihydrogen citrate. In some embodiments, this molar excess is from about5 to about 10,000 fold; in particular about 8 to about 10,000 fold. Itis believed that an excess of citric acid over silver dihydrogen citrateof greater than about 5, more particularly greater than about 10, andeven more particularly greater than about 12, provides substantialstability of the silver dihydrogen citrate complex in solution. Thus,the invention provides solid silver dihydrogen citrate compositionscomprising silver dihydrogen citrate and greater than about 5-, inparticular at least about 8-, more particularly at least about 10-, andeven more particularly at least about 12-fold molar excess of citricacid over silver dihydrogen citrate.

It has surprisingly been found that anhydrous silver dihydrogen citratecompositions are capable of being reconstituted to produce anantimicrobially active composition. It has not been previously reportedthat an anhydrous silver dihydrogen citrate composition would dissolvein a diluent without disrupting the association between the silvercation and the citrate anion; nor has it been shown that reconstitutedsolutions have antimicrobial properties.

The invention provides reconstituted silver dihydrogen citrate andcitric acid compositions containing from about 0.05 ppm to about 10,000ppm of silver ion (Ag⁺). An effective concentration of silver dihydrogencitrate depends on the microbe against which protection is sought, thesubstrate to be treated and the existing or potential bioburden on thesubstrate, and presence or absence of additional antimicrobial activesin the diluent. In some embodiments, an antimicrobially effectiveconcentration of silver ion ranges from about 0.075 ppm to about 2,500ppm, especially about 0.1 to about 1,000 ppm.

Thus, the invention provides a method of using an anhydrous silverdihydrogen citrate composition produce a reconstituted silver dihydrogencitrate solution. The method comprises combining the anhydrouscomposition with a diluent. The diluent is a solvent capable ofdissolving the silver dihydrogen citrate and citric acid. In someembodiments, a suitable diluent is an aqueous solvent, such as water ora solution comprising water and an additional water-soluble ingredient.For example the diluent can be water, ethanol or a combination of waterand ethanol, such as a 10% solution of ethanol in water.

The invention further provides for reconstituting the anhydrous silverdihydrogen citrate and citric acid compositions with a diluent that issubstantially pure water. The term “substantially pure water” meanswater that is essentially free of contaminants or antimicrobial agents,such as ethanol. Examples of substantially pure water are distilledwater, double distilled water, double distilled deionized water,ultrafiltered water and water for injection.

The invention further provides for reconstituting the anhydrous silverdihydrogen citrate and citric acid compositions with tap water, with“fresh water,” that is water obtained from rivers, lakes, streams,reservoirs, etc., and with “treated water,” that is effluent water thathas been treated to remove contaminants. In most normal circumstances,normal tap water will suffice as a diluent, although a purer form ofwater may be used if necessary.

The invention also provides for reconstituting the anhydrous silverdihydrogen citrate and citric acid compositions with a diluent that isan aqueous solution comprising water and another ingredient. The otheringredient can be a cleaning agent, an antibacterial agent or anothertype of ingredient. Exemplary cleaning agents include detergents.Exemplary antimicrobials include ethanol. The invention providesdiluents comprising water and a member of the group consisting ofdetergents, alcohols and combinations thereof. Exemplary diluentsinclude aqueous ethanol, aqueous sodium dodecyl sulfate, and aqueousmixtures of ethanol and sodium dodecyl sulfate. For example, theinvention provides as a diluent an aqueous solution of about 0.1 toabout 10% ethanol and optionally about 0.0001 to about 0.1% detergent.An exemplary diluent comprises about 2% ethanol and greater than about0.01% detergent in water.

The combination of silver dihydrogen citrate and ethanol producesunexpected synergistic disinfectant and anti-microbial properties. Ascompared to an aqueous ethanol composition without silver dihydrogencitrate, the invention provides disinfectant and antimicrobial effectsat a lower concentration of alcohol than is necessary without the silverdihydrogen citrate.

The invention further provides methods of using anhydrous silverdihydrogen citrate compositions to produce an antimicrobial effect. Themethods include combining an anhydrous silver dihydrogen citratecomposition with a diluent to produce a reconstituted silver dihydrogencitrate solution. The methods further comprise applying thereconstituted silver dihydrogen citrate and citric acid solution to asubstrate. The term “substrate” is generically used herein to mean anysurface, article or environment that is in need of antimicrobialtreatment.

Various surfaces may be treated with compositions comprising silverdihydrogen citrate, including countertops, floors, glass surfaces, metalsurfaces (such as stainless steel, chrome and copper surfaces), tilesurfaces, concrete surfaces, vinyl flooring and painted surfaces. Theterm “surface” is used herein to connote any surface, including interiorand exterior surfaces of various objects, including interiors ofcontainers (such as boilers, water tanks, swimming pools, etc.),interiors of pipes, exteriors of household fixtures and appliances,countertops, glass windows and doors. The term “surface” is used hereinto distinguish over a whole article. The reconstituted silver dihydrogencitrate and citric acid solutions are applied to the surface by in aconventional manner, such as by pouring, spraying or swabbing thesolution onto the surface. The solution are conveniently wiped or rinsedoff the surface, or are advantageously left on the surface to dry,thereby providing a long-lasting antimicrobial residue or film on thesurface.

The invention further includes methods of treating various articles,such as fabrics, metal articles, plastic articles, natural products andother articles that are often treated with aqueous cleaning solutions.For example, the invention provides for treating food items with areconstituted silver dihydrogen citrate and citric acid composition.Exemplary food items that are treated in such a manner includevegetables and fruits. Exemplary vegetables that may be treated in thismanner include: roots (such as carrots, beets, radishes); tubers (suchas potatoes, turnips, sweet potatoes and yams); bulbs (such as onions,scallions); corms (such as garlic); green leafy vegetables (such asspinach, kale, lettuce and cabbage); cruciferous sprouts (such asbroccoli and cauliflower); and legumes (such as beans and peanuts).Exemplary fruits that may be treated in this manner include: squash,melons, apples, peaches, pears, bananas, tomatoes, citrus (such asoranges, grapefruit, tangerines, tangelos, lemons and limes), grapes andolives. The invention provides for spraying or wiping the silverdihydrogen citrate composition onto the food item. The inventionalternatively provides for dipping the food item into a the solution.

The invention also embraces methods of treating a variety of articleswith reconstituted silver dihydrogen citrate solutions. For example, thesolutions may be sprayed or wiped onto a variety of items includingmechanical parts used in food service or food process manufacturing. Thesolutions may also be used to clean toys and other items handled bychildren and infants. The solutions may further be used to clean fabricitems, such as clothing, wash rags, bedding and other fabric items. Theinvention provides reconstituted silver dihydrogen citrate compositionsas soaking compositions for disinfecting cooking utensils, eatingutensils and cutlery.

The invention further includes antimicrobially effective silverdihydrogen citrate compositions. The term “antimicrobially effective”means that the composition is effective to halt or reduce the spread ofone or more microbes, prevent infestation by one or more microbes, killone or more microbes, or a combination of these effects. The termmicrobe includes a bacterium, virus or fungus. Aqueous silver dihydrogencitrate compositions have been shown to be effective against a varietyof indicator and pathogenic microbes, including bacteria, such as:Pseudomonas aeruginosa, Salmonella choleraesuis, Staphylococcus aureus,Proprionibacterium acnes, Escherichia coli, Listeria monocytogenes andEnterococcus faecium; viruses such as: Human immunodeficiency virus 1(HIV 1), Herpes simplex virus 2 (HSV 2), Influenza A, Rhinovirus andPoliovirus type 2; and fungi such as Trichophyton mentagrophytes.

The invention may be more fully appreciated with reference to thefollowing illustrative and non-limiting examples. Other embodiments ofthe invention may be practiced within the scope of the presentinvention.

EXAMPLES Example 1 Preparation of Stock Silver Dihydrogen CitrateSolution

Water was introduced into a reverse osmosis unit, passing through asemi-permeable membrane to remove impurities and producing deionizedwater. Anhydrous 99% pure citric acid was mixed with the water toproduce 200 gallons of a 20% (wt/vol) (796 g citric acid per gallonwater) solution. The 200 gallons of 20% citric acid were directed intoan ion chamber containing having positive and negative electrodes, eachconsisting of 200 troy ounces of 999 fine silver. The positive andnegative electrodes were spaced at least 2.0 mm apart, allowing thecitric acid solution to pass between the two electrodes. An iongeneration controller (IGC) power supply including a positive and anegative conductor was attached to the positive and negative electrodes.The IGC applied a current of 5 amps at 17 volts, pulsed every 9 seconds,with a polarity change at 1 minute intervals. Throughout the process,the electrode gap was adjusted in order to maintain the 5 amp-17 voltoutput. The electric current flow caused an ion current to flow betweenthe positive and negative electrodes, producing free silver ions withinthe diluted citric acid solution. The silver ions reacted with thecitric acid in the citric acid solution to produce the silver dihydrogencitrate solution. The 20% citric acid solution was recirculated throughthe ion chamber at 50 gallons per minute for 144 hours until the desiredsilver ion concentration was obtained. The silver dihydrogen citratesolution was then allowed to sit in order to allow any solids formedduring the procedure to precipitate. The resulting product was a silverdihydrogen citrate solution having a silver ion concentration of 2400ppm. Hereafter, this solution is referred to as a stock solution. Thestock solution can be used immediately per the following examples orstored for later use.

It should be understood by those skilled in the art that numerousvariations in the size and/or spacing of the electrodes and numerousvariations in the peak voltage and numerous variations in the timingsequence of the intermittent voltage polarity can readily be used toobtain the silver dihydrogen citrate for use in the invention.

Example 2 Preparation of Anhydrous Silver Dihydrogen Citrate

A 1,000 ml sample of the stock solution, prepared as in foregoingExample 1, was obtained from SSA Batch No. 04.06.03 and was confirmed tohave a concentration of ionic silver of 2400 ppm using an Orion™ 290Aprocessor with an ion specific electrode (ISE). The processor wascalibrated using Orion™ standard solutions.

The 1,000 ml stock solution was decanted into two 2.5″×8″×15″ Pyrex™heat resistant glass trays. The liquid stock solution level wasapproximately 1.5″ in each tray.

The trays were then placed into a small commercial-grade freeze-dryingunit, which consisted of a drying chamber with temperature controlshelves, a condenser to trap water removed from the product, a coolingsystem to supply refrigerant to the temperature control shelves andcondenser, a vacuum system to reduce the pressure in the chamber andcondenser to facilitate the drying process. Cooling and vacuum pressuresettings were kept within the standard ranges associated withfreeze-drying aqueous solutions >50% water matrix.

After 99% of the water was removed from the product, the large, coarsecrystals were collected from the trays and ground into a finecrystalline powder, weighed and stored at an ambient room temperature(74° F.) for one week. Total crystal weight was 206.12 g.

Example 3 Reconstitution of Silver Dihydrogen Citrate Solution

The crystalline powder from Example 2 was then reconstituted usingapproximately 794 ml of pharmaceutical-grade pure water in a 1,000 mlPyrex glass flask. The solution was then agitated via magnetic stirrerfor 30 minutes and put into a light-proof storage cabinet for 24 hours.

The concentration of silver ion in the reconstituted solution was thenmeasured using the Orion 290A processor and ISE described in Example 2.Measurements were obtained at 24, 28 and 72 hours and at intervals of 1week for a total of 4 weeks. The 24 hour reading and the week 4 readingswere 2398 ppm and 2407 ppm, respectively. These concentrations areessentially the same as the 2400 ppm stock solution (within the % erroroperational specifications for the Orion 290A™ processor).

Example 4 Antimicrobial Efficacy of Reconstituted Silver DihydrogenCitrate Solutions

In order to demonstrate the efficacy of silver dihydrogen citratecompositions as preservatives, stock solutions of silver dihydrogencitrate, as described in Example 1, were subjected to PreservativeChallenge Tests. The Preservative Challenge Tests were performedaccording to the European Pharmacopoeia test method 4.04/5.01.03.00 forCategory 2 products (topically used products made with aqueous bases orvehicles, nonsterile nasal products, emulsions including those appliedto mucous membranes.

Bacterial test organisms and yeasts were cultivated on Casein Soymealpeptone agar and fungal test organisms on Sabouraud 4% glucose agar for18-24 hours at 35° C. (bacteria), 48 hours at 25° C. (Candida) or 1 weekat 25° C. (Aspergillus).

After incubation, the bacterial and yeasts were harvested by washing offthe surface of the agar plates with 0.9% sodium chloride. Aspergilluswas harvested by washing off the agar plate surface with 0.9% sodiumchloride/0.01% Tween 80.

The suspension of test microorganisms were diluted with 0.9% sodiumchloride to the final test organism suspensions with a density of −10⁸colony forming units.

Per test organisms, 20 g of the test product were weight in glass jars(250 ml jars with screw cups from Schott/Germany) and contaminated with0.2 ml of the test organism suspension. The microorganisms werecarefully distributed in the test product by stirring with a glassspatula.

The so contaminated test products were stored at 20-25° C. in the dark.

Samples of 1 g material were taken immediately after contamination ofthe test products and 2 days, 7 days, 14 days and 28 days aftercontamination.

The samples were diluted in 0.9% sodium chloride and 0.1 ml aliquots ofthe dilutions were spread on agar plates by means of Drigalsky spatula.An adequate inactivator (neutralizer) of the specific antimicrobial wasincorporated in the diluent used for preparation of the productdilutions and in the agar plates used for assessment of the total numberof viable cells.

The agar plates were incubated for 24 hours at 35° C. (bacteria andyeasts) or 3 days at 25° C. (Aspergillus) and the grown colonies werecounted after the incubation phase. The colonies were counted and thenumber of viable cells (colony forming units) per g test product wascalculated. The log reduction of the microorganisms in the product wasthen calculated (see tables with results of Preservation Challenge Testsbelow).

Test strains:

Pseudomonas aeruginosa ATCC 9027; NCIMB 8626; CIP 82.118

Staphylococcus aureus ATCC 6538; NCTC 10788; NCIMB 9518; CIP 4.83

Candida albicans ATCC 10231; NCPF 3179; IP 48.72

Aspergillus niger ATCC 16404; IMI 149007; IP 1431.83

Silver dihydrogen citrate was tested in a variety of formulations forits antimicrobial effects. The following Tables 1-3 show the results ofthese tests: TABLE 1 Preservative Challenge Test/Deodorant Emulsion Testorganisms Staph. aureus E. coli Ps. aeruginosa C. albicans A. niger O/WPK03-260-01 (Placebo)  2 days after contamination <100 <100 <100 6.0 ×10E4 1.8 × 10E5  7 days after contamination <100 <100 <100 1.6 × 10E43.0 × 10E5 14 days after contamination <100 <100 <100 8.8 × 10E3 2.8 ×10E5 28 days after contamination <100 <100 <100 1.4 × 10E3 n.d. O/WPK03-260-01 (0.1% Axenohl)  2 days after contamination <100 <100 <100<100 1.6 × 10E5  7 days after contamination <100 <100 <100 <100 2.6 ×10E5 14 days after contamination <100 <100 <100 <100 1.2 × 10E5 28 daysafter contamination <100 <100 <100 <100 n.d. O/W PK03-260-01 (0.3%Axenohl)  2 days after contamination <100 <100 <100 <100 1.4 × 10E5  7days after contamination <100 <100 <100 <100 1.2 × 10E5 14 days aftercontamination <100 <100 <100 <100 1.0 × 10E5 28 days after contamination<100 <100 <100 <100 n.d.

TABLE 2 Preservative Challenge Test/Deodorant Emulsion Test organismsStaph. aureus E. coli Ps. aeruginosa C. albicans A. niger PK03-262-01(Placebo)  2 days after contamination 2.0 × 10E4 1.0 × 10E5 1.0 × 10E61.0 × 10E6 2.0 × 10E5  7 days after contamination <100 3.1 × 10E3 4.4 ×105   6.2 × 10E5 3.0 × 10E5 14 days after contamination <100 1.0 × 10E21.1 × 10E6 1.3 × 10E6 3.5 × 10E5 28 days after contamination <100 1.2 ×10E2 6.0 × 10E7 1.1 × 10E6 n.d. PK03-262-01 (0.1% Axenohl)  2 days aftercontamination 5.0 × 10E2 4.0 × 10E2 3.0 × 10E2 4.0 × 10E3 1.4 × 10E5  7days after contamination <100 2.0 × 10E2 <100 3.4 × 10E4 1.8 × 10E5 14days after contamination <100 <100 <100 1.1 × 10E5 2.0 × 10E5 28 daysafter contamination <100 <100 <100 4.0 × 10E5 n.d. PK03-262-01 (0.3%Axenohl)  2 days after contamination 3.0 × 10E2 4.0 × 10E3 7.0 × 10E24.0 × 10E3 1.8 × 10E5  7 days after contamination <100 2.0 × 10E2 <1002.4 × 10E3 2.4 × 10E5 14 days after contamination <100   100 <100 1.2 ×10E3 2.4 × 10E5 28 days after contamination <100 <100 <100 2.0 × 10E2n.d.

TABLE 3 Preservative Challenge Test/Shower Gel Test organisms Staph.aureus E. coli Ps. aeruginosa C. albicans A. niger FB02-060-03 (Placebo) 2 days after contamination 4.0 × 10E2 2.2 × 10E5 in progress 2.2 × 10E52.4 × 10E5  7 days after contamination <100 1.1 × 10E5 in progress 2.1 ×10E5 3.6 × 10E5 14 days after contamination <100 8.2 × 10E4 in progress9.0 × 10E4 3.6 × 10E5 28 days after contamination n.d. n.d. in progressn.d. n.d. Body Shampoo (0.1% Axenohl)  2 days after contamination <1001.8 × 10E4 <100 <100 2.0 × 10E5  7 days after contamination <100 <100<100 <100 3.0 × 10E5 14 days after contamination <100 <100 <100 <100 3.2× 10E5 28 days after contamination <100 <100 <100 <100 n.d. Body Shampoo(0.3% Axenohl)  2 days after contamination <100 6.0 × 10E3 <100 <100 1.8× 10E5  7 days after contamination <100 <100 <100 <100 2.4 × 10E5 14days after contamination <100 <100 <100 <100 2.4 × 10E5 28 days aftercontamination <100 <100 <100 <100 n.d.

The invention may be more fully appreciated with reference to thefollowing illustrative and non-limiting examples. Other embodiments ofthe invention may be practiced within the scope of the invention.

1. A composition comprising anhydrous silver dihydrogen citrate andcitric acid.
 2. The anhydrous composition of claim 1, wherein thecomposition is substantially free of water.
 3. The anhydrous compositionof claim 1, wherein the composition contains less than about 0.5% water.4. The anhydrous composition of claim 1, wherein the composition iscrystalline.
 5. The anhydrous composition of claim 1, wherein thecomposition comprises a molar excess of citric acid over silverdihydrogen citrate.
 6. The anhydrous composition of claim 1, wherein thecomposition comprises at least a 5 fold molar excess of citric acid oversilver dihydrogen citrate.
 7. A process of making a compositioncomprising an anhydrous silver dihydrogen citrate and citric acid,comprising the steps of: (a) providing a stock solution comprisingsilver dihydrogen citrate, citric acid and water; (b) freezing the stocksolution to form a frozen solution; and (c) freeze-drying the frozensolution to form the anhydrous composition.
 8. The process of claim 7,wherein the citric acid is a molar excess of citric acid over silverdihydrogen citrate.
 9. The process of claim 7, wherein step (c) furthercomprises heating the frozen solution.
 10. A method of using ananhydrous silver dihydrogen citrate and citric acid composition,comprising combining the anhydrous silver dihydrogen citrate compositionwith an amount of aqueous diluent sufficient to produce a reconstitutedantimicrobially active silver dihydrogen citrate composition.
 11. Themethod of claim 10, wherein the aqueous diluent is substantially purewater.
 12. The method of claim 10, wherein the aqueous diluent compriseswater and at least one member of the group consisting of alcohols anddetergents.
 13. A method of using an anhydrous silver dihydrogen citrateand citric acid composition, comprising combining the anhydrous silverdihydrogen citrate composition with sufficient aqueous diluent toproduce an antimicrobially effective silver dihydrogen citrate solution;and applying the antimicrobially effective silver dihydrogen citratesolution to a substrate, whereby an antimicrobial effect is achieved.14. The method of claim 13, wherein the substrate is a solid surface, afood article or a non-food article.
 15. The method of claim 14, whereinthe solid surface is the interior of a pipe, a holding tank, a swimmingpool, a spa, a cooling system or a cooling tower.
 16. The method ofclaim 13, wherein the antimicrobial effect is biocidal or biofilmcontrol.
 17. The method of claim 13, wherein the antimicrobial effect isfood preservation.